Haemotoxilyn and Eosin: Two of the most common tissue stains in pathology are haemotoxilyn and eosin. FIG. 1 displays the spectral transmission of tissue components stained with haemotoxilyn and eosin. The data of FIG. 1 is from the following article: Bautista, Abe, Yamaguchi, Yagi and Ohyama, “Digital staining of pathological tissue specimens using spectral transmittance,” Proc. of SPIE Vol. 5747, 1892-1903 (2005).
Color Human Vision: Color vision in humans is typically described with a small set of spectral lines. The relevant spectral lines for the invention disclosed herein are as follow: g (436 nm), F (486 nm), e (555 nm), d (588 nm), and C (656 nm). The relative sensitivities of human vision at these lines are roughly: 7% at g, 24% at F, 100% at e, 68% at d, and 5% at C.
Human vision has poor spatial resolution at blue wavelengths. The fovea of the retina contains very few blue cones while the smaller foveola contains none. Human vision is largely based upon the red and green cones. These cones are commonly designated by wavelength as short (blue), middle (green) and long (red).
In a typical optical design for human vision, the F (486 nm), d (588 nm), and C (656 nm) spectral lines are specified. These three lines include a small portion of the blue range (440-490 nm). The g (436 nm) line is frequently ignored due to lack of blue sensitivity and blue resolution in human vision.
Color Machine Vision: Color machine vision is defined by the spectrum of the source and sensitivity of the detector. FIG. 2 displays the relative color sensitivity of the combination of a white LED and a color CCD. The LED is a CBT-90 by Luminus Devices, Inc. of Billerica, Mass., USA. The CCD is a KAI-08050 by the Eastman Kodak Company of Rochester, N.Y., USA.
A CCD sensor has large sensitivity at both the g and F lines. The quantum efficiencies of the color KAI-08050 sensor are: 38% at the g line, 45% at the F line, 20% at the d line, and 30% at the C line. The blue channel is more sensitive than the green or red.
A typical white LED comprises a blue gallium-nitride LED with phosphor coating. The emission spectrum comprises a peak at 450 nm from the LED, and broad phosphorescence from 450 nm to 700 nm.
The typical dyes of pathology are haemotoxilyn and eosin. The typical dyes of pathology create images with strong components at the g and F lines.
Correction of the g-line in film or CCD sensors requires the use of special glass. A combination of long crown and short-flint is common in objective lenses for both photography and microscopy.
There are two common formats of color vision by CCD sensors. A first format of color vision employs three CCD sensors and two beamsplitters. Consistent orientation of the images to the CCDs requires precise alignment of optical hardware, which is difficult and expensive. A shifted or rotated image requires interpolation for conversion to the correct location and angle. The sampling rate is defined by the width of a single pixel. A second format of color vision employs a Bayer pixel which comprises 1 blue pixel, 2 green pixels, and 1 red pixel. The image sampling rate of a Bayer is defined by the width of 2 pixels.
Glass Types: The refractive index of glass is defined by dipole current. A dipole comprises positive and negative charge with finite separation. The electric field of a dipole counters the external field of the electromagnetic wave. The opposing electric field of the dipole drives the total electric field back to zero faster than outside of the glass. Thus, the dipole current shortens the spatial wavelength. The effects of dipole current upon wavelength are represented by refractive index.
The growth in refractive index within a wavelength range is described by dispersion. The Abbe number defines dispersion as
            v      d        =                            n          d                -        1                              n          F                -                  n          C                      ,
wherein n specifies the refractive index according to spectral lines. Partial dispersion defines the growth in refractive index of a first range as a fraction of growth in refractive index of a second range. An example of partial dispersion is
      P    gF    =                              n          g                -                  n          F                                      n          F                -                  n          C                      .  
The normal partial dispersion of glass types is defined by Schott North America, Inc. of Elmsford, N.Y., USA (hereinafter “Schott”) asPgFn=+(0.6438)−(0.001682)νd.
The relative partial dispersion is defined by a departure from the normal partial dispersion as below.ΔPgF=PgF−PgFn 
A crown glass employs tightly bound electrons with a resonant frequency in the near ultraviolet range. Consequently, a crown glass displays a refractive index that grows faster at shorter wavelengths. A crown glass displays a normal dispersion with nearly zero relative partial dispersion. Examples of crown glass are borosilicate and fused silica. A long crown employs tightly bound electrons with a resonant frequency in the deep ultraviolet. Consequently, the refractive index is smaller than crown glass, and the dispersion is very small as represented by a large Abbe number. The relative partial dispersion of a long crown is positive. The “long spectrum” is due to a large spectral separation of absorption peaks. Examples of long crown are fluorophosphate glass and calcium fluoride crystal. Both materials are more expensive and more difficult to process than crown glass.
A short flint employs defects that reduce the lifetime of dipole oscillations. Consequently, the amplitude of the dipole current grows less rapidly while approaching resonance, and the partial dispersion is reduced. The relative partial dispersion of a short flint is negative. Consequently, a short flint may counter the partial dispersion of a crown. The reduced dipole current also indicates a broadening of the ultraviolet absorption band. This broadened absorption band shortens the spectral length between absorption bands. The broadened ultraviolet absorption band might penetrate the blue spectrum. A broadened spectrum of the infrared band has no effect upon the red spectrum. Examples of short flint are lead borate, and niobium silicate. A recently developed short flint is zirconium-doped tantalum silicate.
A lanthanum flint employs the tightly bound electrons of a rare earth metal. The high atomic number of a rare earth metal provides a much larger electron density than a long crown. A lanthanum flint typically displays a negative relative partial dispersion.
A dense crown provides a higher refractive index than a crown or long crown. The higher refractive index can reduce astigmatism and spherical aberration. A dense crown typically displays a positive relative partial dispersion.
A dense flint provides a higher refractive index than a crown or long crown. The higher refractive index can reduce astigmatism and spherical aberration. A dense flint displays increasing dispersion at shorter wavelengths. Consequently, the benefits of a dense flint often create more lateral color in the blue spectra.
Recent developments of short flints were motivated by ecological concerns about lead borate. The use of niobium, tantalum, and zirconium has created new short flints with superior qualities. In particular, N-KSFS11 by Schott employs zirconium-doped tantalum oxide within a largely silica glass network. The tantalum oxide provides a higher refractive index than past short flints. The zirconium doping shortens the resonant lifetime of the tantalum oxide. Other vendors such as Ohara Inc. of Japan (hereinafter “Ohara”) did not employ zirconium doped tantalum oxide as of 2011. Thus, N-KSFS11 was unique to Schott North America, Inc, at least as of 2011.
CCD Size: A KAI-08050 sensor by the Eastman Kodak Company of Rochester, N.Y., USA (hereinafter “Kodak”) employs four CCDs on a single chip. The sensor diagonal is 22.66 mm. Each sub-sensor has its own external circuitry. A large CCD is desired for rapid mapping of a large specimen such as a tissue specimen. However, the large angular size of the KAI-08050 detector occupies a compromised portion of the field of tube lens in the prior art. Thus, implementation of a larger CCD with 25 mm diagonal has been limited by the quality of the tube lens. In particular, the lateral color of tube lens is compromised at field margin. Astigmatism and Petzval curvature of the tube lens are also common at large diagonals of 25 mm.
20× Objective Lens: A popular objective lens for pathology is an apochromat, such as part number UPLSAPO 20×, by Olympus America, Inc. of Center Valley, Pa., (hereinafter “Olympus”) at 20× at 0.75 NA. Herein, the vision NA is defined by the objective NA. The brightness of the image background is determined by the 0.75 NA of vision when the NA of illumination is larger. However, the resolution is not determined by 0.75 NA of vision. The typical cover strata of a tissue specimen create spherical aberration beyond the correction of the objective lens. Consequently, the diffraction limit of the objective is circa 0.50 NA. The annular NA of vision from 0.50 to 0.75 provides space for an illumination lens stop at 0.60 NA. A properly designed vision lens-stop at 0.50 NA can stop the illumination at 0.60 NA to 0.50 NA without scatter by the inside faces of the glass elements. A sharp edge to aperture defines a proper design for a vision lens stop. The effective NA of illumination at 0.50 can precisely match the NA of vision without background fog from scatter.
The measured densities of glass components of the Olympus UPLSAPO 20× indicate fluorophosphate and niobium silicate as two of the glass types. The fluorophosphate is a long crown, and the short flint is a niobium silicate. Thus, the apochromat at 20× at 0.75 NA includes long crown and short flint as glass types.
Critical Illumination: In 1875, Nelson empirically determined that an optical system is spatially incoherent when the illumination NA exceeds 0.75 times the vision NA. This condition defines the “critical illumination” of Nelson. In practice of the current invention, an illumination NA at 0.80 times the vision NA creates a small edge overshoot which is highly beneficial to image quality. The edge overshoot is created by a negative partial coherence between adjacent point spreads of the objective lens. The partial coherence of the illumination field is defined by the Fourier transform of the angular profile of the source. Thus, a nominal range for Nelson's critical illumination is defined as an illumination NA at 0.75-0.85 times the vision NA. Nelson employed an image of the source at the object of illumination. Consequently, numerous authors erroneously indicate “critical illumination” as a source image located at the object. Thus, “critical illumination” must be defined by the relation of illumination NA to objective NA.
Kohler Illumination: In 1893, Kohler defined an illumination system with the source image at a great distance from the object of illumination. Typically, the distant image of the source is created by location of a source image at the front focal point of the condenser lens. However, the lens stop of the objective lens is frequently not located at the back focal point of the objective. Thus, the location of the source conjugate must be carefully considered.
A Kohler illumination system is defined by Seward in Optical design of microscopes published by SPIE Publications in 2010. There, the illumination system has a source, a source lens, an illumination field stop, an illumination lens stop, a condenser lens, and an illumination field. The Kohler illumination system creates a distant image of the source from the illumination field.
Bistable Telescope: There are two common formats of a refractive telescope: a Keplerian, and a Galilean. A Keplerian telescope employs an objective lens and ocular lens, both with positive power. A Galilean telescope employs an ocular lens with negative power. A Galilean telescope is shorter than a Keplerian telescope at the same magnification. The reduction in length between Galilean and Keplerian telescopes is twice the effective focal length of the ocular lens. The shorter path length of a Galilean telescope is preferred in the tube of a microscope.
The prior art contains examples of telescopes within microscopes. For example, U.S. Pat. No. 4,195,903 to Kawase describes a rotatable Galilean telescope within the tube region of an infinity-corrected microscope. In U.S. Pat. No. 4,673,973, Ledley describes a Galilean telescope within one arm of a split optical system.
The bistable telescope is related to numerous patents. In U.S. Pat. No. 3,804,506, for example, Fletcher describes a camera shutter with rotary solenoids operative to actuate shutter blades. An inertial damper and a stop plate are built into each solenoid to prevent shock and rebounding.
With U.S. Pat. No. 4,262,989, Waters teaches changeable sets of Galilean optics mounted on a rotary shaft that is rotated by a rotary solenoid. Three magnifications are achieved by a single Galilean telescope at three orientations: forward along the optic axis, backward along the optic axis, and across the optic axis. The shaft and the optics are fixed at angular dispositions by engagement of a detent ball with grooves in a detent plate.
U.S. Pat. No. 7,245,425 to Miyashita is directed to an optical system with an objective lens and an intermediate magnification varying part in the form of a telescope. The telescope rotates by 180 degrees to reverse direction. At magnification of 1.4×, the telescope achieves a 2× change in magnification between modes. The system also includes a reduction stop in one mode.
Nyquist Parameters: The cut-off frequency of a diffraction-limited objective lens is
      f    CO    =                    2        ⁢                                  ⁢        NA            λ        .  
At 0.588 um, the cutoff frequency for the diffraction-limited 0.50 NA objective is 1.70 cycles per um. The Nyquist rate is the minimum sampling rate at which there is no overlap of the replicated spectra. The replicated spectra are centered at the harmonic frequencies of the sampling wave form. An overlap between replicated spectra creates aliasing. The Nyquist rate at 0.50 NA and 0.588 um wavelength is 3.4 cycles per um. FIGS. 3A and 3B display examples of replicated spectra with and without aliasing.
In FIG. 3A, an 11.00 um pixel and 20× magnification defines the sampling frequency as 1.82 cycles per um. There is significant aliasing from the replicated spectrum. The optical transfer function (OTF) of the irradiance is defined as the addition of the replicated spectrum to the original spectrum. Thus, the OTF is artificially large near the cut-off frequency at 1.70 cycles per um. The magnitude of the replicated spectrum is defined by the duty cycle of the pixel sampling pattern. Thus, the magnitude of the replicated is smaller than unity. At any magnitude of the replicated spectrum, the aliasing creates an artificially large OTF near the cut-off frequency at 1.70 cycles per um.
In FIG. 3B, an 11.00 um pixel and 40× magnification defines the sampling frequency as 3.64 cycles per um. There is no aliasing from the replicated spectrum. Thus, the optical transfer function (OTF) is a true description of the optical system up to the onset of replicated spectrum. The original spectrum may extend to one-half of the sampling frequency without aliasing. This condition lays the foundation for the Nyquist frequency, which has been defined as one-half of the sampling frequency. The Nyquist frequency represents the largest spatial frequency that can be sampled without aliasing. In a system with an 11.00 um pixel, the Nyquist frequency is 0.91 cycles per um at 20× as FIG. 3A, and 1.82 cycles per um at 40× as in FIG. 3B. The Nyquist frequency at 40× exceeds the cut-off frequency at 1.70 cycles per urn, while the Nyquist frequency at 20× does not.
The Nyquist parameters of the current invention are summarized in Table 1 below. The 11.0 um pixel is defined by use of the Bayer pixel of a Kodak KAI-08050 as referenced hereinabove, which has a pixel size of 5.5 μm. Clearly, the 40× mode satisfies both Nyquist conditions for sampling without aliasing, while the 20× does not. A 0.50 NA microscope with 11.0 um Bayer pixel displays aliasing. Thus, a 2× telescope is essential for the elimination of aliasing in a typical 20× microscope for pathology.
TABLE 1Nyquist parameters of the 20X and 40X magnificationsParameterValuePixel5.50 μmBayer pixel11.00 μmProjected Bayer pixel at 20X0.55 μmProjected Bayer pixel at 40X0.28 μmCut-off frequency (588 nm, 0.50 NA)1.70 cyc/μmNyquist rate (2X cut-off fequency)3.40 cyc/μmSampling frequency at 20X1.82 cyc/μmSampling frequency at 40X3.64 cyc/μmNyquist frequency at 20X0.91 cyc/μmNyquist frequency at 40X1.82 cyc/μm